DNA is susceptible to a variety of mutations and chemical modifications. Errors during DNA replication, either mispairing or slippage, result in mismatched base pairs, which occur at a frequency of 10-8 to 10-6. Exposure to UV irradiation or chemical agents may lead to covalently modified DNA bases, and programmed meiotic and mitotic DNA rearrangement, ionizing radiation and oxidative agents can result in double-strand DNA breaks. To maintain genomic integrity and to sustain life, bacteria, archaea and eukarya use conserved mechanisms to repair or to tolerate each type of damage. My research group has continued to carry on structural and functional studies of E. coli and human mismatch repair processes and lesion-bypass DNA synthesis.[unreadable] [unreadable] Mismatch repair (MMR) in E. coli is initiated by three proteins, MutS, MutL and MutH, to specifically target the newly synthesized daughter strand. MutS is an ATPase and recognizes a mismatched base-pair as well as an insertion or deletion of 1-4 nucleotides in one strand. MutH is a latent endonuclease that is both sequence- and methylation-specific; when activated by MutS upon detection of a mismatch, it cleaves 5? to the unmethylated d(GATC) sequence in a hemimethylated duplex. MutL mediates the communication between MutS and MutH, which do not directly interact. Once a nick is introduced to the daughter strand by MutH, UvrD helicase, single-strand binding protein and DNA exonuclease are recruited to remove nucleotides from the nick to beyond the mismatch. Homologues of MutS and MutL are found in all eukaryotes, and malfunction of either human MutS or MutL homolog is directly implicated in the susceptibility to hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. Our previous studies led to the determination of crystal structures of MutS, MutS-mismatch DNA and MutS-mismatch-ADP complexes, the N- and C-terminal domain of MutL, and finally MutH and MutH-DNA complexes and biochemical characterization of the role of the MutS and MutL ATPases and the cleavage specificity of MutH. In the currrent year, we have succeeded in determining the crystal structure of UvrD helicase-DNA complexes, which represnt the consecutive physical steps of UvrD unwinding a duplex DNA in an ATP hydrolysis cycle (manuscript is being reviewed, Lee JY & Yang W).[unreadable] [unreadable] My group has sepnt nearly 4 years on the structural characterization of a Y-family DNA polymerases, which perform low-fidelity synthesis on undamaged DNA templates and are able to traverse normally replication-blocking lesions, including abasic sites, 8-oxo-G, benzopyrene adducts, and cyclobutane pyrimidine dimers. Y-family polymerases are widespread and enable species from E. coli to human to tolerate UV irradiation and various forms of base modification. We and others found that the active site of Y-family polymerases are larger and more solvent exposed than that of replicative (high fidelity) polymerases, which explain their unique ability in translesion synthesis. A nagging question is why the Y-family polymerases have much better fidelity (accuracy in template-dependent DNA synthesis) than predicted from merely base:base hydrogen bonding. After publishing the first Y-family polymerase and DNA complex structure in 2001 and a serier of crystal structures of Dpo4 complexed with a cyclobutane pyrimidine dimers, benzo[a]pyrene adduct, and abasic lesion in 2003 and 2004, last year we capped our studies of Dpo4 by determining its substrate specificity and nucleotide selection (Vaisman et al., 2005). Our structural and biochemical studies suggest that both replicative and translesion DNA polymerase depend on precise metal-ion coordination for the rate-limiting step ? the chemical bond formation. The latest study prompted us to take a close look on the two metal ions in the active site essential for the polymerase activity. In conjunction with our studies of sequence and structure-specific nucleases, MutH and RNase H, we conclude that the requirement of two Mg2+ ions for nucleic acid substrate binding and phosphoryl transfer reaction greatly enhances the catalytic specificity of the polymerases and nucleases (Nowotny & Yang, EMBO, 2006; Yang et al., Mol Cell, 2006).[unreadable] [unreadable] We are continuing our efforts in characterizing the role of RAG1 and RGA2 in V(D)J recombination. Most recently, our pursue has led down the path of studying histone modification as V(D)J recombination is dependent on transcription activation. RAG2 contains a PHD domain, which is recently shown to bind trimethylated Lys4 of histone H3. We have determined the crystal structures of the RAG2 PHD domain along and complexed with the modified H3 peptide. We are in the process of preparing the manuscript.[unreadable] [unreadable] [unreadable] [unreadable]